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Currently, our knowledge of how different cell types in a tissue microenvironment respond to low and high linear energy transfer (LET) radiation is highly restricted. In this study, a comparative analysis was performed on γ-ray-induced DNA damage and repair in primary human melanocytes and keratinocytes isolated from 3 donors. Our study demonstrates a modest interindividual variability in both melanocytes and keratinocytes in terms of both spontaneous and ionizing radiation (IR)-induced 53BP1 foci formation and persistence. Melanocytes, in general, showed a slightly elevated (1.66-2.79 folds more) 53BP1 foci induction relative to keratinocytes after exposure to different doses of γ-rays (0.1-2.5 Gy) radiation. To verify the influence of ATM kinase on IR-induced 53BP1 foci formation, melanocytes and keratinocytes were treated with a specific ATM kinase inhibitor (KU55993, 10 μM) for 1 h prior to radiation. ATM kinase inhibition resulted in the reduction of both spontaneous and IR-induced 53BP1 foci by 17-42% in both melanocytes and keratinocytes of all the 3 donors. Increased persistence of IR-induced 53BP1 foci number was observed in ATM-inhibited melanocytes and keratinocytes after different post exposure times (6 h and 24 h). Taken together, our study suggests that interindividual variations exist in the induction and repair of DNA double-strand breaks (DSBs) in melanocytes and keratinocytes and that ATM is crucial for an optimal DSB repair efficiency in both human skin cell types., (© 2022 S. Karger AG, Basel.)

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 ± 2 days old) yielding approximately 30 x 10 6 viable cells per mouse. Primary adult mouse keratinocytes are harvested from the dorsal skin of female mice. Male mice (~6 weeks old) can be used for keratinocyte harvesting depending on the requirements of the experiment. Euthanized mice are shaved and sterilized with serial washes in povidone iodine and ethanol solutions (70% alcohol). After disinfecting the mice, the dorsal skin is removed and the subcutaneous fat and muscle are removed with a scalpel and discarded. The skins are cut into small pieces and treated with a mild, low temperature trypsinization to detach the lower dermis from the epidermis. The scraped epidermises are stirred at low speed, filtered to remove the hairs, counted, and re-suspended in culture medium. This method provides an excellent single cell suspension of highly culturable cells for many downstream applications.

Skin lipids (e.g., fatty acids) are essential for normal skin functions. Epidermal FABP (E-FABP) is the predominant FABP expressed in skin epidermis. However, the role of E-FABP in skin homeostasis and pathology remains largely unknown. Herein, we utilized the 7,12-dimethylbenz(a)anthracene and 12-O-tetradecanolyphorbol-13-acetate-induced skin tumorigenesis model to assess the role of E-FABP in chemical-induced skin tumorigenesis. Compared to their wild-type littermates, mice deficient in E-FABP, but not adipose FABP, developed more skin tumors with higher incidence. 12-O-tetradecanolyphorbol-13-acetate functioning as a tumor promoter induced E-FABP expression and initiated extensive flaring inflammation in skin. Interestingly, 12-O-tetradecanolyphorbol-13-acetate -induced production of IFN-β and IFN-λ in the skin tissue was dependent on E-FABP expression. Further protein and gene expression arrays demonstrated that E-FABP was critical in enhancing IFN-induced p53 responses and in suppressing SOX2 expression in keratinocytes. Thus, E-FABP expression in skin suppresses chemical-induced skin tumorigenesis through regulation of IFN/p53/SOX2 pathway. Collectively, our data suggest an unknown function of E-FABP in prevention of skin tumor development, and offer E-FABP as a therapeutic target for improving skin innate immunity in chemical-induced skin tumor prevention., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Charruyer and colleagues (this issue) report two significant advances to the field of cutaneous keratinocyte stem cells: a pair of new selectable markers that recognize a subset of α6(+)CD34(+) label-retaining cells, and an in vivo limiting dilution assay for keratinocyte stem cells with long-term repopulating ability. This work has important implications for keratinocyte stem cell identification and assay, as well as for the identification of target cells in non-melanoma skin cancer.

The mammalian skin is a complex dynamic organ composed of thin multilayered epidermis and a thick underlying connective tissue layer dermis. The epidermis undergoes continuous renewal throughout life. The stems cells uniquely express particular surface markers utilized for their identification, isolation and localization in specific niches in epidermis as well as hair follicles (HFs). The two stage skin carcinogenesis model involves stepwise accumulation of genetic alterations and ultimately leading to malignancy. Whereas early research on skin carcinogenesis focused on the molecular nature of carcinogens and tumor promoters, more recent studies have focused on the identification of the target cells and tumor promoting cells for both chemical and physical carcinogens and promoters. Recent studies support the hypothesis that keratinocyte stem cells are the targets in skin carcinogenesis. In this review, we discuss briefly the localization of stem cells in the epidermis and HFs, and review the possibility that skin papillomas and carcinomas are derived from stem cells, as well as from other cells in the cutaneous epithelium whose stem cell properties are not well known., (© 2012 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2012 The American Society of Photobiology.)

The skin provides an anatomical barrier to physical, chemical and biological agents. Hence, it is not surprising that it has well-developed innate immunity. What we find surprising is that the CD49f(+) /CD34(+) hair follicle stem cells should have an enriched expression profile of so many genes involved in innate immunity. Do these stem cells require extra protection from environmental insults? Or, could there be a new role for these genes? To probe these questions, we first summarize the roles of some key players in epidermal innate immunity. We next focus on their expression in CD49f(+) /CD34(+) hair follicle stem cells. Then, we consider recent data suggesting a new role for these 'old players' in the regulation and mobilization of haematopoietic and mesenchymal stem cells. Finally, we hypothesize that the 'old players' in these hair follicle stem cells may be playing a 'new game'., (© 2012 John Wiley & Sons A/S.)

Understanding keratinocyte stem cell regulation is important in understanding the pathogenesis of wound healing and nonmelanoma skin cancer. We previously used a sensitive and quantitative assay for in vitro keratinocyte colony formation and mapped the keratinocyte stem cell locus (Ksc1) on mouse chromosome 9. Examination of the candidate genes in this locus disclosed a sequence variant in the gene for bone morphogenetic protein 5 (Bmp5). In this report, we used a naturally occurring mouse with a null mutation in this gene to probe stem cell properties in mouse epidermis. We found that the mutant keratinocytes had a significant reduction in the size and number of clonogenic keratinocytes. The mutant mice had a 50% reduction in the number of label-retaining cells when compared with their littermates. Addition of exogenous Bmp5 protein increased the number and size of keratinocyte colonies in the mutant as well as their wild-type littermates. Surprisingly, the mutant mice showed at least a 2-fold increase in skin tumor susceptibility over their littermates. We conclude that a naturally occurring mutation in Bmp5 affects keratinocyte stem cell proliferation, and skin tumor susceptibility, and is a candidate stem cell regulatory gene in the Ksc1 locus.

Although ideas on the stem cell origins of cancer date more than one hundred years, critical evidence to support these theories is largely lacking. Our objective here is to outline our historical perspective on keratinocyte stem cells in the cutaneous epithelium and to summarize specific evidence suggesting that epithelial stem cells may contribute to chemically induced skin cancer. We note that, while strong evidence does support this hypothesis, experiments in progress may provide direct visualization of tumors derived from hair follicle stem cells.

Primary adult mouse keratinocytes are harvested from the dorsal section of 6- to 8-wk-old mice. Euthanized mice are clipped and sterilized with serial washes in provodine iodine and ethanol solutions. The skin of the mice is removed and treated with trypsin to detach the dermis from the epidermis. Cells harvested from this proven method can be used for molecular biology, biochemistry, or a variety of cell culture techniques, including the clonogenic cell assay presented here.

To identify slowly cycling (label-retaining) keratinocytes in the cutaneous epithelium of mice, newborn female pups were injected with [3H]-thymidine twice daily for 3 d beginning the third day after birth. At 8-10 wk of age, skin from the dorsal side of the mice was removed and fixed. The skin was processed for paraffin embedding, and serial sections were prepared. Dewaxed and ethanol-treated slides were dried and dipped into autoradiographic emulsion and allowed to dry in light tight box for 4 wk at 4 degrees C. After fixation with photographic fixer, the slides were washed, lightly stained, and viewed with a light microscope to observe and quantify cells that retained the labeling. Label-retaining cells were identified by the black silver grains over their nuclei.

Skin cancers as seen in the clinic are the result of a long history of events of which only the final stages are easily observed. As normal cells progress to the neoplastic and later metastatic stages, multiple changes in gene expression and cellular phenotypes occur. Nevertheless, the early events in the pathway leading from the first exposure to carcinogenic or mutagenic agents to a frank tumor are thought to involve a two-step process of tumor initiation and tumor promotion. In experimental two-stage skin carcinogenesis in mice, benign and malignant neoplasms can be induced on the backs of mice following a low, or sub-threshold, exposure to a carcinogen (initiation) and subsequent chronic regenerative epidermal hyperplasia caused by a variety of physical, chemical, or biological agents (promotion). Tumor initiation is thought to involve conversion of some of the epidermal cells into latent neoplastic cells, whereas promotion elicits expression of the neoplastic change. Many questions remain about this process, in particular the identity and biological properties of the cells that are specifically the targets of tumor initiation and promotion. Conceivably, any proliferative cell could become and remain initiated; however, these rare cells in the cutaneous epithelium able to become neoplastic cells after exposure to carcinogens and tumor promoters have many of the properties of stem cells. Although this concept that stem cells are the target cells in the development of cancer is not new, I will consider here the evidence that the target cells are indeed stem cells in the cutaneous epithelium.

Mouse keratinocyte stem cells originate from the bulge of hair follicle, and, according to definition, possess a clonogenic activity in vitro. We have investigated seven inbred (C57BL/6, C3H, DBA/2, BALB/c, FVB) and outbred (SENCAR, CD-1) mouse strains and found that three genetically distinct subsets of mouse strains differ significantly in the frequency of clonogenic activity in vitro. The analysis of keratinocyte colonies in two reciprocal backcross [C57BL/6 x (BALB/c x C57BL/6); BALB/c x (BALB/c x C57BL/6)] and intercross [(BALB/c x C57BL/ 6)F2] of BALB/c and C57BL/6 mice allowed us to identify two subpopulations of clonogenic keratinocytes able to produce small (less than 2 mm2) and large (more than 2 mm2) colonies. We conducted linkage analysis and found that small colonies associated with mouse chromosomes 1, 6, 7, 8, and 9; but large colonies--with the chromosome 4. We defined locus on the chromosome 9 that associated with small colonies as keratinocyte stem cell locus 1 (Ksc1), and locus on the mouse chromosome 4 associated with large colonies-keratinocyte stem cell locus 2 (Ksc2). Ksc1 and loci on chromosomes 6 and 7 are close if not equal to loci associated with sensitivity to skin carcinogenesis. We conclude that two subpopulations of stem cells able to produce small and large colonies regulated by different genes and genes regulating small colonies might be responsible for sensitivity to skin carcinogenesis.

It is widely believed that epithelial stem cells reside in the hair follicle bulge region. We investigated the hematopoietic stem and progenitor cell marker, CD34, as a potential marker of hair follicle bulge keratinocytes. Using a CD34-specific antibody, we identified intense membrane staining on keratinocytes in the bulge region of the mouse hair follicle. CD34 expression colocalized with both slowly cycling (label retaining) cells and keratin 15 expression. Live CD34+ keratinocytes were positively selected using antibodies to CD34 and alpha6 integrin in combination with fluorescent activated cell sorting. Sorted cells were analyzed for DNA content, and a staining profile was generated to confirm these cells as keratinocytes. CD34+ keratinocytes were predominantly in Go/G1, in contrast to CD34- cells, which had well defined G2/M and S phases. In addition, CD34+ keratinocytes were found to express alpha6 integrin more intensely than CD34- cells (p<0.05), identifying this population as an alpha6 integrin bright subset. When seeded at clonal density, CD34+ keratinocytes formed larger colonies than CD34- cells (p<0.05), indicating a higher proliferative potential. All flow-sorted cells were positive for keratin 14 expression, and negative for keratin 1, loricrin, vimentin, and CD31. The majority of CD34+ cells (98%) were positive for keratin 6, establishing this population as basal keratinocytes of follicular origin. CD34 message was detected by reverse transcription polymerase chain reaction predominantly in the CD34+ keratinocytes, confirming specificity of the antibody. This work is the first to demonstrate that CD34 is a specific marker of bulge cell keratinocytes in the cutaneous epithelium. Furthermore, the use of this marker facilitates isolation of live epithelial cells with stem and progenitor cell characteristics, potentially providing a tool for the study of carcinogen target cells, gene therapy, and tissue engineering applications.

Several converging lines of evidence suggest that keratinocyte stem cells including those clonogenic in vitro, are the target cells in skin carcinogenesis. However, the mechanism of their involvement in carcinogenesis continues to be unknown. Recently, we have demonstrated that the number of keratinocyte clonogenic stem cells from mice is a genetically defined and quantitative complex trait. In this study, we analyzed the size of the colonies in BALB/c, C57BL/6, and their (BALB/cxC57BL/6)F1 hybrid mice. We found that the size of the colonies is also genetically regulated and that there are at least two types of clonogenic keratinocytes that form either small or large colonies. Small and large colony numbers were significantly different in BALB/c and C57BL/6 mice. We performed a genome-wide scan of small and large colony number in the two backcrosses [BALB/cx (BALB/cxC57BL/6)F1]; [C57BL/6x (BALB/cxC57BL/6) F1]; and [(BALB/cxC57BL/6)F1x (BALB/cxC57BL/6)F1] intercross mice. We report here that the loci on chromosomes 4 and 9 play a role in the regulation of large and small colony number respectively. We conclude that mouse epidermis has two major types of stem cells generating small and large colonies, and regulated by different genes. Our results suggest that the loci, including one major and several minor loci linked to the subpopulation of keratinocytes forming small colonies were related, if not identical to, the loci linked to susceptibility or resistance to skin tumor promotion and skin tumor development.

We tested five inbred strains and two outbred stocks of female mice in a quantitative assay for clonogenic keratinocytes from the cutaneous epithelium. We found three significantly different subsets of colony counts such that: C57BL/6 C3H = DBA/2 = SENCAR = BALB/c > FVB = CD(-1) in culture conditions optimized for CD(-1) 0. C57BL/6 and BALB/c, two inbred parental strains, were chosen for further analysis. The F1 generation of these two parental strains had an intermediate number of colonies. The keratinocyte colony number from the two backcross generations was significantly different, while the colony number in the F2 generation was intermediate between the two backcrosses. We conclude that the number of keratinocyte colonies represents a new genetically definable quantitative trait. Analysis suggests that this trait is multigenic where the genes have an additive but not necessarily equal effect. We have therefore laid the foundation for identifying these stem cell regulatory genes, which may provide a new perspective on the mechanism of carcinogenesis and a new target for gene therapy.

Background: Kruppel-like factor KLF4 is a transcription factor critical for the establishment of the barrier function of the skin. Its function in stem cell biology has been recently recognized. Previous studies have revealed that hair follicle stem cells contribute to cutaneous wound healing. However, expression of KLF4 in hair follicle stem cells and the importance of such expression in cutaneous wound healing have not been investigated. Methodology/Principal Findings: Quantitative real time polymerase chain reaction (RT-PCR) analysis showed higher KLF4 expression in hair follicle stem cell-enriched mouse skin keratinocytes than that in control keratinocytes. We generated KLF4 promoter-driven enhanced green fluorescence protein (KLF4/EGFP) transgenic mice and tamoxifen-inducible KLF4 knockout mice by crossing KLF4 promoter-driven Cre recombinase fused with tamoxifen-inducible estrogen receptor (KLF4/ CreERTM) transgenic mice with KLF4(flox) mice. KLF4/EGFP cells purified from dorsal skin keratinocytes of KLF4/EGFP transgenic mice were co-localized with 5-bromo-2'-deoxyuridine (BrdU)-label retaining cells by flow cytometric analysis and immunohistochemistry. Lineage tracing was performed in the context of cutaneous wound healing, using KLF4/CreERTM and Rosa26RLacZ double transgenic mice, to examine the involvement of KLF4 in wound healing. We found that KLF4 expressing cells were likely derived from bulge stem cells. In addition, KLF4 expressing multipotent cells migrated to the wound and contributed to the wound healing. After knocking out KLF4 by tamoxifen induction of KLF4/CreERTM and KLF4(flox) double transgenic mice, we found that the population of bulge stem cell-enriched population was decreased, which was accompanied by significantly delayed cutaneous wound healing. Consistently, KLF4 knockdown by KLF4-specific small hairpin RNA in human A431 epidermoid carcinoma cells decreased the stem cell population and was accompanied by compromised cell migration. Conclusions/Significance: KLF4 expression in mouse hair bulge stem cells plays an important role in cutaneous wound healing. These findings may enable future development of KLF4-based therapeutic strategies aimed at accelerating cutaneous wound closure. [ABSTRACT FROM AUTHOR]